Volume   Journal

International
I Number 1 2010 for Environmental Rehabilitation and Conservation
[17-21]
Volume I No. 1 2010 [17-21] Zade[ISSN
et al. 0975 - 6272] 

Toxicity testing of industrial effluents through freshwater fish Lebistes

reticulates (Peters)

Suresh B. Zade1, Shashikant R. Sitre2, Shanta Satyanarayan3 and Sanjeev S. Gandhewar4

Received: February 24, 2009 ⏐ Accepted: May 01, 2010 ⏐ Online: April 4, 2010

Abstract
Toxic effects of industrial effluents of an herbal
pharmaceutical company to freshwater fish
Lebistes reticulatus (Peters) were investigated
during 96 hours static bioassay tests. LC50
values for raw, neutralized and physico-
chemically treated industrial effluents of an
herbal pharmaceutical manufacturing company
were found out under standard laboratory
conditions. The behavior of the fish is also
recorded during experiments. It is evident from
the toxicological studies that raw industrial
effluent was much more toxic as compared to
neutralized and physico- chemically
treated effluent and toxicity was reduced by
more than 30% with physico-chemical
treatment alone. In order to further know the
reduction in toxic effect the-physico-
chemically treated industrial effluent was
subjected to biological treatment by activated
sludge system. The effluent after biological
treatment revealed no toxic effect to Lebistes
reticulatus for about a month pointing out that
toxicity was fully reduced after biological
treatment and the wastewater can be discharged
into inland surface waters without harming the
aquatic biota.

Keywords: Acute toxicity ⏐ Lebistes Introduction

reticulates ⏐ industrial effluent ⏐ Activated
Today, herbal medicines are popular in India
sludge
and some of the south East Asian Countries due
to very low side effects, cultural acceptability
and low cost (Rajashekharan, 2002). The herbal
For Correspondence: medicines are manufactured from plant
materials like roots, stems, barks, gums, resins
1
P.G. Department of Zoology, RTM Nagpur University, and certain chemicals like sugars, gums,
Nagpur, Maharashtra organic solvents, gelatin, lactose, salts, special
2
N.S. Science and Arts College, Bhadrawati, District – minerals, various heavy metals etc.
Chandrapur, Maharashtra
3
National environmental engineering research Institute Herbal medicines generate a lot of waste water
(NEERI), Nagpur, Maharashtra (effluent) during manufacturing processes
4
J.B. College of Science, Wardha, Maharashtra
which include washings of medicinal plants to

Toxicity testing of industrial effluents through freshwater fish Lebistes reticulates (Peters)
17

Volume I Number 1 2010 [17-21]
Zade et al.
remove dust, dirt and microbial contaminants.
Apart from general washing the wastewater is
generated from different processes like
crushing, mixing, extraction, distillation,
fermentation, decoction and utensil washing
based on market demand (Vanerkar et al.,
2002). The organic, inorganic and toxic
components present in the effluent have direct
impact on aquatic organisms and it is very
difficult to correlate the observed effect to
specific pollutants as these effluents are
complex in nature having highly fluctuating
characteristics. Due to fluctuating market
demands these medicines are prepared in
batches and so the characteristics of effluent are
continuously changing. These wastewaters
have high Biochemical Oxygen Demand
(BOD), Chemical Oxygen Demand (COD) and
very high Suspended Solids (S.S.) with large
amount of heavy metals, phenolics etc.
Materials and Methods
The static bioassay tests were performed at
room temperature using Lebistes reticulates as
test organism. The required fishes for
experimentation purpose were procured from a
local fish supplier of Nagpur City (Maharshtra
State). The length of fish ranged between 1.5 to
2.0 cm. having a weight of 0.20 to 0.30 gms.
approximately. Healthy fish were selected for
toxicity testing experiments.
The industrial effluents were collected from a
local herbal pharmaceutical manufacturing
company from Nagpur and composite sample
were collected and utilized for toxicity testing.
The raw, neutralized and physico-chemically
treated effluents were characterized as per
Standard Methods (APHA, 1998) and their
characteristics are presented in Table 1. For
experimentation standard dilution water was
prepared and used, Methods for measuring the
Toxicity testing of industrial effluents through freshwater fish Lebistes reticulates (Peters)
toxicity of industrial effluents were followed as
per standard protocols (Doudorof, 1951,
Sprague, 1969, Rao et al.,1982),
The bioassay studies were carried out in glass
aquaria of 10 Lit capacity using ten fishes in
each container. Similar control was run parallel
with dilution water only. Suitable
concentrations were prepared and a range
finding test and final confirmatory test were
performed and readings on fish mortality were
recorded at every 24 hours interval.
LC50 values were calculated as per Litchfield
and Wilcoxon (1949). The results were
subjected to statistical analysis. Based on LC50
values, 95% confidence limits, slope and
regression (R2) are calculated and presented in
Table 2. Fish behaviour was also studied during
the experiment of 96 hours duration.
Results and Discussion
The toxicity studies revealed that raw and
neutralized wastewaters were much more toxic
to Lebistes as compared to physico-chemically
treated effluents. The fishes came to surface
frequently due to distress in raw wastewater,
with quick opercular movements and moving
erratically. The distress to fishes is due to
reduction in dissolved oxygen by raw
wastewater because of high Biochemical
Oxygen Demand (BOD) values and its acidic
nature. Somewhat lessened effect was observed
in other two wastewaters. Loss of balance of
the fish was observed at higher concentration of
wastewater. The LC50 values (Table 2) clearly
show that raw wastewater was much more toxic
and neutralized effluent toxicity was reduced
somewhat due to marginal reduction of BOD,
COD and Suspended Solids with slight removal
of heavy metals.
18
  
Volume I Number 1 2010 [17-21]
Zade et al.

It is thus inferred that this wastewater needs completely treated effluent is now non toxic to
treatment to reduce toxicity and lime fish as shown by healthy fish.
neutralization is effective in reducing the The herbal pharmaceuticals though age old and
toxicity. So the wastewater was subjected to referred in our Vedic scripts did not receive
physicochemical treatment using conventional much attention till recently but its importance
coagulants like ferric chloride, alum, lime and was understood quite late and they gained
ferrous sulphate. The cationic polymer popularity due to low side effects. Moreover
Oxyfloc-FL 11 gives good results at a dose of being herbal in nature their toxicity testing
300:0.25 mg/ liter at optimum combination for aspect was not seriously considered so far and
reduction in toxicity of the effluent. It was so they were not fully understood.
found that the toxicity was reduced by more
than 30%. Still it is not safe to discharge the Today bioassays provide a safe tool for
effluent as fishes do not survive for a long assessing the toxic effects of industrial
duration into it. So the effluent was subjected to wastewaters and chemicals and LC50 values
further biological treatment by aerobic play an important role in protecting the fish
activated sludge system and toxicity tests were communities (Basak and Konar 1977). The
conducted. results obtained from the bioassays will help
the industries to take necessary pollution
It was confirmed from the toxicity tests that the control measures before discharging effluents
effluent was completely safe for discharge after into natural waters which will help in
biological treatment as no fish mortality was minimizing the pollution and safeguarding our
observed for a period of one month. The aquatic organisms.
Parameters Raw effluent Neutralized Physic-chemically
effluent treated effluent
Colour Dark Yellow Grey Light Yellow
pH 3.6-4.00 6.9-7.50 6.6
Total acidity 1385 590 192
Total suspended solid 1800 1603 295
Total solids 4169 2536 538
BOD (5 days at 20°C) 6892 4820 1660
COD 12430 9600 3860 Table 1: Characteristics of
Sulphide as S-2 28 20 09 Herbal Pharmaceutical
Total Phosphates (PO4- 136 98 42 Industrial Effluents
2)

Total Nitrogen 223 132 60

Oil and Grease 82 36 15
Heavy Metals Note:
Iron 34.40 15.85 8.20
All the values are expressed in
Mercury 0.015 0.006 0.002
mg/Litre except colour and pH.
Copper 0.5790 0.312 0.222
Manganese 3.540 1.100 0.169 The heavy metals were
Zinc 0274 0.1624 0.100 analyzed on atomic absorption
Nickel 0.808 0.28 0.142 spectrophotometer.
Lead 1.562 0.92 0.72
Chromium 0.2330 0.1204 0.0721
Selenium 0.211 0.131 0.095
Arsenic N.D. N.D. N.D.
Toxicity testing of industrial effluents through freshwater fish Lebistes reticulates (Peters)
19
  
Volume I Number 1 2010 [17-21]
Zade et al.

Time 
Parameter  Raw effluent  Neutralized  Physico‐ chemically 
in Hrs  effluent  treated effluent 
24  LC50  6.50  7.50 38.00
95%  4.2 ‐ 10.7   5.22‐10.90   33.2‐43.00 
hrs 
Confidence  y=  0.0613x  +  y=0.04812x+  Y=0.057x + 35.40  Table  2:  LC50  and  Other 
Limit, Slope  3.60  5.166  0.989 
Estimated  Values  of  Acute 
R2  0.962  0.966 
48  LC50  6.00  7.00 Toxicity  Tests  for  Lebistes 
37.50 
95%  3.05‐11.50   5.60‐8.75  
hrs  33.8‐42.2   reticulates  (Peters)  Exposed  to 
Confidence  Y= 0.0623x +  Y= 0.0413x + 
Y= 0.055x + 35.08 
Limit, Slope  2.90  4.936  Industrial  Effluents  (Herbal 
0.984 
R2  0.979  0.974 
72  LC50  4.90  6.70 Pharmaceutical)
37.30 
95%  2.70‐8.80   4.82‐9.10  
hrs  33‐41.00  
Confidence  Y=  0.0625x+  Y=  0.0405x+ 
Y= 0.057x+ 34.6 
Limit, Slope  1. 794  4.612 
0.2785 
R2  0.978  0.972 
96  LC50  2.45‐7.80  6.30
36.80 
95%  2.45‐7.80   4.25‐9.30  
hrs  33.50‐40.20  
Confidence  Y=  0.0558x+  Y=  0.041  x+ 
Y= 0.06Ix+ 33.90 
Limit, Slope  1.597  4.261 
0.975 
R2  0.982  0.983 
 

References

APHA (1998). Standard Methods for the guppy Lebistes reticulates (Peters). Bull. Environ.
Examination of Water and Wastewater, 20th Contam.Toxicol. 54: 57-59.
Edition, APHA, AWWA and WEF,
Litchfield J.T and Wilcoxon T.A. (1949).
Washington D.C.
Simplified Method of evaluating dose
Basak, P.K. and Konar S.K.(1977). A new method effect experiment. J. Pharmacol. Exper.
for the determination of safe concentration Theory 96:99-113.
of insecticides to protect fishes, Indian J
Rajashekharan P.E. (2002). World of Science:
.Environ. Health, 19(4): 283-292.
Herbal Medicine, Ministry of Information
Doudoroff, P., Anderson, E.G., Burdick, G.E., and Broadcasting, New Delhi, Employment
Galtsaf P.S., Hart W.E., Patrick, E.R., News, Sept. 21-27, pg.3-4.
Strong, E.R., Surber, E.W. and Vanitron,
Rao, DMR, Jothi Kumar and Krishnamoorthi K.P.
W.M. (1951). Bioassay Methods for the
(1982). A Course Manual on Fish Bioassay,
evaluation of acute toxicity of industrial
National Environmental Engineering
wastewater to fish. Sewage Ind. Waste, 23:
Research Institute, (NEERI), Nagpur. 1-53.
130.
Shrinivas, P, Benarji G, and Rao P. (1994).
Kumar, S., Sahay, S.S. and Sinha M. K. (1995).
Preliminary observation on the effect of
Bioassay of distillery effluent on common

Toxicity testing of industrial effluents through freshwater fish Lebistes reticulates (Peters)
20
  
Volume I Number 1 2010 [17-21]
Zade et al.

industrial effluents to certain animals. Sprague, J.B. (1969). Measurement 0 Pollutant

Journal of Environ. Health. 26(1): 57-59.
Stephan, S. Raj M.A., Murugesan A.G. and Hanifa
M.A.(1987). Toxicity of industrial effluents
to the freshwater catfish Mystus keletius,
Current Science 56 (14): 733-735. 10.
Sangli, A.B, and Kanabur V.V. (2002). Acute
toxicity of chloro-aniline and pentachloro-
phenol to freshwater fish Gambusia affinis.
Environ.& Ecol. 18(1): 78-80.
Toxicity to Fish - I: Bioassay Methods for
Acute Toxicity, Water Research, 3 : 7 – 52.
Vanerkar, A.P, Satyanarayan Shanta, Dharmadhikri
D.M. and Kaul S.N. (2002): Application of
synthetic polyelectrolytes (Anionic/ Cationic)
for treatment of herbal pharmaceutical waste
water. Proc. Indo-Italian Workshop, NEERI,
Nagpur, Sept. 2-4.

Toxicity testing of industrial effluents through freshwater fish Lebistes reticulates (Peters)
21